The study of biology has recently benefited from improved methods of analysis and sequencing of nucleic acids. While the “human genome” has been sequenced there are still vast amounts of genomic material to analyze, e.g., genetic variation between different individuals, tissues, additional species, etc.
Devices for DNA sequencing based on separation of fragments of differing length were first developed in the 1980s, and have been commercially available for a number of years. A number of new DNA sequencing technologies are based on the massively parallel analysis of unamplified (WO00006770; Proceedings of the National Academy of Sciences U.S.A, 100, 3960-3964 (2003)) or amplified single molecules, either in the form of planar arrays (WO9844151) or on beads (WO04069849; Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005); Nat Biotechnol. 6, 630-6344 (2000)).
The methodology used to analyze the sequence of the nucleic acids in such new sequencing techniques is often based on the detection of fluorescent nucleotides or oligonucleotides. The detection instrumentation used to read the fluorescence signals on such arrays may be based on either epifluorescence or total internal reflection microscopy, for example as described in WO9641011, WO00006770 or WO02072892.
Multiplexing enables large sample numbers to be simultaneously sequenced during a single experiment. Some methodologies utilize individual “barcode” sequences that are added to each sample so that they may be differentiated during data analysis.